High density lipoprotein (HDL), its main protein, apolipoprotein A-I (apoA-I), and mimetics of apoA-I have been shown in a number of laboratories to reduce inflammation in animal models of disease (Getz and Reardon (2011) J. Inflamm. Res. 4: 83-92; Navab et al. (2012) Arterioscler. Thromb. Vasc. Biol. 32: 2553-2560; Degoma and Rader (2011) Nat. Rev. Cardiol. 8: 266-277; Yao et al. (2012) Front. Pharmacol., 3: 37; Navab et al. (2010) Arterioscler. Thromb. Vasc. Biol. 30: 164-168).
In particular, the use of such ApoA-I mimetic peptides such as 4F to modulate diseases has been demonstrated in a wide variety of contexts including, but not limited to animal models of arthritis (Charles-Schoeman (2008) Clin. Immunol. 127: 234-244) asthma (Nandedkar et al. (2011) J. Lipid Res., 52: 499-508) atherosclerosis (Navab et al. (2011) J. Lipid Res. 52: 1200-1210), Alzheimer's disease (Handattu et al. (2009) Neurobiol. Dis. 34: 525-534), cancer (Su et al. (2010) Proc. Natl. Acad. Sci. USA, 107: 19997-20002; Gao et al. (2011) Integr. Biol. (Camb). 3: 479-489; Ganaphthy et al. (2012) Int. J. Cancer, 130: 1071-1081), diabetes (Morgantini et al. (2010) Diabetes. 59: 3223-3228), hepatic fibrosis (DeLeve et al. (2008) Am. J. Pathol. 173: 993-1001), kidney disease (Vaziri et al. (2009) Kidney Int. 76: 437-444; Vaziri et al. (2010) Nephrol. Dial. Transplant. 25: 3525-3534), obesity (Peterson et al. (2009) J. Lipid Res. 50: 1293-1304), osteoporosis (Sage et al. (2011) J. Bone Miner. Res. 26: 1197-1206), scleroderma (Weihrauch et al. (2007) Am. J Physiol. Heart Circ. Physiol. 293: H1432-H1441), systemic lupus erythematosus (Woo et al. (2010) Arthritis Res. Ther., 12: R93), transplant vasculopathy (Hsieh et al. (2007) Transplantation 27:84:238-243), and vascular dementia (Buga et al. (2006) J. Lipid Res. 47: 2148-2160). Thus, the potential benefit of such peptides is great.
The apoA-I mimetic peptide 4F showed great promise in a variety of mouse models of disease (Navab et al. (2010) Arterioscler. Thromb. Vasc. Biol. 30: 164-168) leading to a phase I/II study in humans with high risk cardiovascular disease (Bloedon et al. (2008) J. Lipid Res. 49: 1344-1352). In this study the 4F peptide synthesized from all D-amino acids (D-4F) was administered orally at doses that ranged from 0.43-7.14 mg/kg. The resulting plasma peptide levels were low (Cmax 15.9±6.5 ng/mL). Despite these very low plasma levels, doses of 4.3 and 7.14 mg/kg significantly improved the HDL inflammatory index (HII), which is a measure of the ability of a test HDL to inhibit LDL-induced monocyte chemoattractant protein-1 (MCP-1) production by cultured human artery wall cells; doses of 0.43 and 1.43 mg/kg were not effective (Id.). A second clinical trial focused on achieving high plasma peptide levels using low doses (0.042-1.43 mg/kg) of the 4F peptide synthesized from all L-amino acids (L-4F) delivered by intravenous (IV) or subcutaneous (SQ) administration (Watson et al. (2011) J. Lipid Res. 52: 361-373). Very high plasma levels were in fact achieved (e.g., Cmax 3,255±630 ng/mL in the IV study), but there was no improvement in HII (Id.).
To resolve this paradox, new studies were conducted in mice that led to the surprising discovery that the major site of action for the peptide may be in the intestine, even when the peptide is administered SQ (Navab et al. (2011) J. Lipid Res. 52: 1200-1210). Moreover, the dose administered, not the plasma level, was the major determinant of efficacy (Id.). Efficacy was the same at the same dose when the peptide was administered orally or SQ suggesting that in the compartment controlling peptide efficacy, peptide concentrations should be similar; the peptide concentration was similar only in the feces (Id.). In a subsequent study, this compartment was further identified as the small intestine (Navab et al. (2012) J. Lipid Res. 53: 437-445). Additionally, metabolites of arachidonic and linoleic acids in the enterocytes of the small intestine were found to be ˜10-fold higher than in the liver, but the percent reduction in these metabolites after oral 4F peptide administration was significantly greater in the liver compared to the small intestine strongly suggesting that the small intestine is a major site for peptide action (Id.). As a result of these studies (Navab et al. (2011) J. Lipid Res. 52: 1200-1210; Navab et al. (2012) J. Lipid Res. 53: 437-445), it was concluded that doses of peptide ranging between 40-100 mg/kg/day would be required instead of doses of 0.42-1.43 mg/kg/day as was used in the studies of Watson et al. (Watson et al. (2011) J. Lipid Res. 52: 361-373).
The 4F peptide (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2, (SEQ ID NO:1)) has end blocking groups (Ac— and —NH2) that stabilize the class A amphipathic helix and dramatically increase efficacy (Venkatachalapathi et al. (1993) Proteins Structure Function Genet. 15: 349-359; Yancey et al. (1995) Biochemistry. 34: 7955-7965; Datta et al. (2001) J. Lipid Res. 42: 1096-1104; Anantharamaiah et al. (2007) J. Lipid Res., 48: 1915-1923). In unpublished studies in mice, it was found that in the absence of these end groups the 4F peptide is 25,000-fold less effective in vivo. The required end (protecting) groups for 4F and for a number of other apoA-I mimetic peptides (Navab et al. (2010) Arterioscler. Thromb. Vasc. Biol. 30: 164-168) can only be added by chemical synthesis; living organisms cannot be engineered to make a molecule containing these end groups. Thus, the production of peptide for clinical use at these doses would not be practical because of the cost of producing this amount of peptide by solid phase synthesis.